The present invention relates to an improved membrane based biosensor and to a method of improving the performance of membrane based biosensors.
Biosensors based on ion channels or ionophores contained within lipid membranes that are deposited onto metal electrodes, where the ion channels are switched in the presence of analyte molecules have been described in International Patent Application Nos. WO 92/17788, WO 93/215289, WO 94/07593 and U.S. Pat. No. 5,204,239 (the disclosures of which are incorporated herein by reference). As these biosensors rely on changes in ion conduction through the membrane, usually mediated by an ionophore, it is important that there exists an ionic reservoir between the electrode and the lipid membrane. Ideally this ionic reservoir between the electrode and the lipid reservoir is not totally depleted or filled, by conduction through the ionophore, during the course of the measurement cycle. The usual method of measuring the conductance changes is the use of alternating current (AC) impedance spectroscopy. The abovementioned disclosures have shown that good reservoirs can be produced using special linker lipid compounds.
The present inventors have now found that the application of a direct current (dc) potential offset superimposed onto the AC impedance signal can influence the apparent conduction of ions by the ionophore through the membrane. Without wishing to be bound by scientific theory it is believed that this modification of the ionophore conduction occurs through the modulation of the reservoir capacity and improvement in the reservoir homogeneity. This improvement in conduction of ions by the ionophore therefore allows the use of less ionophore which may be useful in producing more sensitive sensor membranes as less analyte is required to switch the ionophore on/off. A negative dc potential applied to the metal electrode has been shown to improve the ion conduction by ionophores, whereas a positive dc potential applied to the metal electrode has been shown to decrease and even negate the apparent conduction of the ionophores through the membrane. This effect is especially noticeable when membranes are formed containing phosphatidyl choline based lipids. The inventor has found that by controlling the dc offset, the reproducibility of the ionophore conduction is greatly improved.
Accordingly, in a first aspect the present invention consists in an improved membrane based biosensor comprising a lipid membrane incorporating ionophores the conductivity of the membrane being dependent on the presence or absence of an analyte, a reference electrode, a sensing electrode onto which is deposited the lipid membrane such that a functional reservoir exists between the lipid membrane and the sensing electrode, the improvement comprising including in the biosensor means to apply a dc electrical potential offset to the sensing electrode relative to the reference electrode.
In a second aspect the present invention consists in an improved method of detecting the presence or absence of an analyte in a sample using a membrane based biosensor comprising a lipid membrane incorporating ionophores the conductivity of the membrane being dependent on the presence or absence of the analyte, a reference electrode, a sensing electrode on to which is deposited the lipid membrane such that a functional reservoir exists between We lipid membrane and the sensing electrode, the improvement comprising applying a dc electrical potential offset to the sensing electrode relative to the reference electrode.
In a third aspect by incorporating ionisable, polarisable, dipolar or otherwise electroactive species within the membrane based biosensor comprising a lipid membrane incorporating ionophores the conductivity of the membrane being dependent on the presence or absence of an analyte. a reference electrode, a sensing electrode onto which is deposited the lipid membrane such that a functional reservoir exists between the lipid membrane and the sensing electrode, the appropriate dc potential can be induced between the sensor electrode and the analyte solution
Although it is envisaged that generally it is preferred to apply a negative potential onto the metal sensor electrode in order to improve the ionophore conduction, it may be useful in some circumstances to apply a positive potential onto the metal sensor electrode thus reducing or negating the apparent ionophore conduction through the membrane.
In a preferred embodiment of the present invention a dc potential of between +500 mV to xe2x88x92500 mV is applied to the sensing electrode.
In a further preferred embodiment the dc offset is produced through the use of a counter electrode where the electrochemical potential between the counter electrode and the sensing electrode produces an electrical potential of between 0 to xe2x88x92500 mV, with the sensing electrode being at the negative potential.
In a preferred embodiment the counter electrode is made from stainless steel.
In a further preferred embodiment the counter electrode is made from titanium.
In a further preferred embodiment the counter electrode is made from silver, gold, platinum, palladium, copper, chromium or molybdenum.
In another preferred embodiment the counter electrode is made from metals that are capable of being deposited in a thin film onto a plastic, glass or silicon substrate, said metals being stable for at least 30 minutes in aqueous solution and sets up the appropriate electrochemical potential relative to the sensing electrode on addition of an aqueous solution.
In a further preferred embodiment of the present invention the counter electrode is an electrochemically neutral metal relative to the sensing electrode and the dc electrical potential of between +500 to xe2x88x92500 mV is created by electronic means.
In a further preferred embodiment of the present invention the counter electrode produces an electrochemical potential relative to the sensing electrode which is enhanced or negated or reversed using a dc electrical potential created by electronic means to give a potential of between +500 toxe2x88x92500 mV.
In yet another preferred embodiment of the present invention, the dc offset potential of the sensing electrode, onto which is deposited the lipid membrane, is controlled using a three terminal measurement, where the impedance measurement is made between the counter electrode and the working electrode which is the sensing electrode and where the dc offset potential is controlled by a reference electrode to be between +500 to xe2x88x92500 mV as required.
The metals used for the counter electrode and the reference electrode in the three terminal measurement may be any of the commonly used metals and electrode combinations commonly used in these measurements as known to those skilled in the art.
In a further preferred embodiment of the present invention the metal used for the sensing electrode is a layer of freshly evaporated or sputtered gold. Alternatively, a freshly cleaned gold surface, which can be produced using plasma etching or ion-beam milling, can be used.
It is further preferred that the first layer of the lipid membrane is produced using the linker lipid shown in FIG. 1, the disulfide of mercaptoacetc acid, linker gramicidin shown in FIG. 2, the membrane spanning lipid (C) and the membrane spanning lipid (D) both shown in FIG. 3.
It is further preferred that the second layer of the lipid membrane is produced from diphytanyl phosphatidyl choline, glycerol diphytanyl ether, shown in FIG. 7. and biotinylated gramicidin shown in FIG. 4.
In a further preferred embodiment the second layer lipid contains at least a proportion of a phosphatidyl choline, or phophatidyl ethanolamine or phosphatidic acid lipid.
In a further preferred embodiment the second layer lipid contains at least a proportion of a charged lipid.
In a further preferred embodiment the lipid membrane is a monolayer.
As will be appreciated by those skilled in the art, if the sensing of an analyte occurs through the swing off or on of an ionophore contained within the lipid sensing membrane on addition of nalyte, then it is possible to monitor this change in conduction by measuring the amount of electrical potential required in order to maintain the membrane conduction value at the initial ungated membrane conduction value. The magnitude and sign of the electrical potential is then related to the amount of analyte present in the sample.
By increasing the signal spectral inhomogeneity the information content in the sign can be increased with the consequent possibility of improved signal to noise. One mechanism for achieving this is to take advantage of the system voltage dependence by applying a non sinusoidal excitation and then analyzing the results by fourier transform in which case the signal information content will be increased due to the cross modulation products in the output.
By automatically selecting a dc potential the sensitivity can be optimized. This may sometimes require the use of a calibrating dose of analyte for each measurement (See Example 2 as a means of minimizing drift.)
The present invention also provides an improved method for detecting response to an analyte in which a signal may derived by altering and monitoring dc bias potential, while analyte is binding to the channels during the biosensor gating event, either to maintain the admittance constant preferably at the frequency for minimum phase or similarly to maintain the phase constant preferably at the frequency for minimum phase.
The present invention further provides an improved method for detect the electrode response to analyte in which the signal response is optimized by automatically altering the dc bias potential to obtain maximum sensitivity or minimum drift.